Mac-1, a granulocyte-monocyte specific differentiation antigen containing polypeptides of 190,000 and 105,000 MW and Mac-2 and Mac-3, monocyte specific antigens of 32,000 and 110,000 MW, respectively, have been identified using rat anti-mouse monoclonal antibodies. All these macrophage differentiation antigens are expressed in much greater quantities on activated macrophages than on monocytes in bone marrow, blood, or spleen. Mac-1 antigen is found on tumoricidal macrophages in the mouse and through cross-reaction on human monocytes and natural killer cells. Monoclonal antibody Fab fragments coupled to Sepharose will be used to isolate large quantities of radiolabeled antigens for chemical characterization. Subunit relationships will be studied by peptide mapping, cross-linking and gel filtration. The presence of functional and catalytic activities and intracellular phosphorylated sites will be investigated. Amino acid and carbohydrate compositions and NH2-terminals of proteolytic fragments and intact polypeptides isolated on a large scale will be determined. Arrangement in the membrane will be determined by digestion of whole cells, inside-out vesicles and purified detergent soluble antigen bound to Fab affinity beads and other procedures such as 32P, vectorial 125I, and photoactive hydrophobic reagent labelling.